Treatment of dna damage related disorders

ABSTRACT

The present invention provides methods and compositions for prophylaxis and treatment of a variety of disorders including DNA damage related disorders, cancer, ischemia, oxidative stress, atherosclerosis, and stroke using a chloroquine compound.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation-in-part of attorney docket29202-710.831 filed May 26 2004, the US national phase of PCT US03/37838filed Nov. 26, 2003, which is a continuation-in-part of 10/351,733 filedJan. 24, 2003 and is also a continuation-in-part of U.S. Ser. No.10/307,077, filed Nov. 27, 2002, all of which are incorporated herein byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made in the course of research sponsored by theNational Institutes of Health (NIH Grant Nos. CA71387). The U.S.government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Cancer is now the second leading cause of death in the United States.Over 1 million new cases of cancer are expected to be diagnosed in 2003and over 500,000 people are expected to die of cancer.

Cancer is typically treated with one or a combination of three-types oftherapies: surgery, radiation, and chemotherapy. Overall costs forcancer, including treatments, were approximately $170 billion dollars in2002. The cancer treatments are not only expensive; they are ineffectivemost of the time and also have many side effects. Hence, there is ademand for more effective cancer prevention and treatment agents, aswell as for the prevention and treatment of DNA damage relatedconditions.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods of prophylaxis of cancer. The methodscomprise administering to a subject at risk of cancer, an effectiveamount of a chloroquine compound, provided the subject has been free ofa localized skin epithelialoma or carcinoma for at least a year beforecommencing administration of the chloroquine compound. Optionally, thesubject has never suffered from a localized skin epithelialoma orcarcinoma basal cell before commencing administration of the chloroquinecompound. Optionally, the subject has never suffered from skin cancer.

In some methods, the subject is monitored for development of a cancerafter administration of the chloroquine compound. Optionally, themonitoring comprising monitoring the subject for a cancer other than alocalized skin epithelialoma or carcinoma basal cell. Optionally, themonitoring comprising taking a sample of a body fluid, or performing ascan of an internal organ.

In some methods, the subject is at risk of cancer by having aprecancerous tissue. In some methods, the subject has a first cancer,and is at risk of a second cancer. In some methods, the subject has acancer and is undergoing radiation therapy to treat the cancer, theradiation therapy putting the subject at risk of a second cancer; andwherein the administration effects prophylaxis of the second cancer. Insome methods, the chloroquine is administered before or during theradiation therapy. In some methods, the subject has a cancer and isundergoing chemotherapy to treat the cancer, the chemotherapy placingthe subject at risk of a second cancer, and wherein the administrationeffects prophylaxis of the second cancer. In some methods, thechloroquine compound is administered before or during the chemotherapy.

In some methods, the subject is at risk of cancer due to a geneticvariation associated with increased risk of cancer. In some methods, thesubject is at risk of cancer due to viral infection. In some methods,the subject is at risk of cancer due to exposure to a carcinogen orirradiation. In some methods, the subject is at risk of cancer due toexposure to X-rays.

In some methods, there is an additional step of determining presence ofa genetic variation in an ATM gene of the subject associated withcancer. In some methods, there is an additional step of administering achemopreventive agent other than the chloroquine compound to thesubject. In some methods there is a further step of determining the riskof cancer in a subject before administering the chloroquine compound.

In some methods, the chloroquine is administered intravenously. In somemethods, the chloroquine is administered orally.

In some methods, the subject is free of diseases of the immune system,infectious diseases, and neurological diseases. In some methods, thesubject is free of psoriasis, malaria, protozoal infections, Alzheimer'sdisease, Parkinson's disease, lupus erythematosus, rheumatism,hypercalcemia, multiple sclerosis, and migraine.

In some methods, the prophylaxis is effective to prevent detectabledevelopment of cancer for at least six months after administering theeffective dosage. In some methods, the administering is performed beforeexposure of the subject to the risk of cancer. In some methods, theadministering is performed at regular intervals for a period of at leastsix months.

In some methods, the chloroquine compound is selected from the groupconsisting of chloroquine, chloroquine phosphate, hydroxychloroquine,chloroquine diphosphate, chloroquine sulphate, hydroxychloroquinesulphate, or enantiomers, derivatives, analogs, metabolites,pharmaceutically acceptable salts, and mixtures thereof. Optionally, thechloroquine compound is chloroquine, chloroquine phosphate orchloroquine diphosphate.

In some methods, the chloroquine compound has a systemic effect. In somemethods, the patient is human and the dosage is 0.05 to 1 mg/kg per day.In some methods, the patient is human and the dosage is 0.2 to 0.6 mg/kgper day. In some methods, the patient has been exposed to a carcinogenor radiation, and the dosage is administered at least on the day ofexposure and the day following exposure. In some methods, the patienthas been exposed to a carcinogen or radiation, and the dosage isadministered at least on the day before the exposure, on the day of theexposure, and at least on the day following the exposure. In somemethods, the patient is human and has genetic susceptible to cancer, andthe dosage is 0.2 to 0.6 mg/kg week. In some methods, the amount of thecompound administered is up to about 10 mg/kg/day. In some methods, theamount of the compound administered is more than about 0.1 mg/kg/day. Insome methods, the amount of the compound administered is less than about50 mg/kg/day. In some methods, the amount of the compound administeredis less than about 10 mg/kg/day. In some methods, the chloroquinecompound is administered more than once a week. In some methods thechloroquine compound is administered daily. In some methods, thechloroquine compound is formulated in a sustained release formulation.In some methods, the patient is human.

The invention further provides methods of therapeutically treatingcancer comprising administering to a subject having a cancer aneffective amount of a chloroquine compound whereby the cancer istherapeutically treated. Optionally, the cancer is other than alocalized skin epithelialoma or carcinoma basal cell. Optionally, thecancer is other than skin cancer. Optionally, the treatment reduces oreliminates further growth of the cancer. Optionally, the treatmentshrinks or eliminates the tumor. Optionally, the treatment inhibitsinvasion of the cancer into tissues of the subject and/or inhibitsmetastasis of the cancer.

Some methods involve a further step of monitoring changes in the cancerresponsive to the administering. Optionally, the monitoring comprisestaking a sample of a body fluid, or performing a scan of an internalorgan. Optionally, the monitoring comprises identifying a geneticvariation in an ATM gene of the subject associated with cancer.Optionally, the methods involve an additional step of administering achemotherapeutic agent other than the chloroquine compound to thesubject. Optionally, the methods involve an additional step ofdetermining presence of the cancer before the administering step.

In some methods, the subject is free of diseases of the immune system,infectious diseases, and neurological diseases.

In some methods, the patient is human and the dosage is 0.05 to 1 mg/kgper week.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Kaplan-Meier survival curve of C57/BL6 mice afterexposure to 8 Gy total body irradiation (TBI). Half of the cohortreceived a dose of chloroquine (dashed line) by either IP injection(1.75 mg/kg or 3.5 mg/kg) or in their drinking water (3.5 mg/kg or 7mg/kg) the day before the TBI. The one mouse which died in thechloroquine-treated group received 1.75 mg/kg by i.p. injection.

FIG. 2 shows that chloroquine treatment enhances survival after TBI byenhancing recovery of hematopoietic progenitor cells. Five mice received3.5 mg/kg chloroquine (C) by i.p. injection 24 and 4 hours prior to TBI(bars with diagonal stripes). Five mice received no chloroquine(stippled bars). Fourteen days after irradiation, the cellularity (openbars) of hematopoietic tissues (spleen, thymus, bone marrow) wasassessed by a blinded observer on a scale of 0-3 with 3 being normalcellularity. The bars represent the average cellularity of the tissuesfrom the 5 mice in each group.

FIG. 3 shows a Kaplan-Meier survival curve of AT mice after exposure to8 Gy TBI. Half of the cohort received a dose of 3.5 mg/kg chloroquine(CHL; dashed line) by i.p. injection 24 and 4 hours prior to the TBI.

FIG. 4 demonstrates that chloroquine treatment prevents the developmentof tumors in Eμ-myc mice. After weaning, a cohort of transgenic miceexpressing the c-myc oncogene were started on chloroquine (CHL) at 7.0mg/kg in the drinking water ((+), solid line). Within 100 days, all ofthe mice with no drug in the water had died of leukemia, while none ofthe cohort of mice on drug had succumbed. The latter group of mice wasthen divided into two groups (timing of this event depicted by heavyarrow), one group of which was taken off of chloroquine ((−), dashedline) and the other group of which was started on i.p. injections of 3.5mg/kg of chloroquine once a week. Within a month, all of the mice takenoff of chloroquine had developed malignancies and all of the mice on theweekly i.p. injections remained tumor-free for months.

FIG. 5 illustrates that chloroquine treatment reduces the development oftumors in mice injected with the potent chemical carcinogen,3-methylcholanthrene (3-MC). Chloroquine (CHL, 3.5 mg/kg) was given byi.p. injection 24 and 4 hours prior to 3-MC injection in 30 mice and 30mice received the carcinogen with no chloroquine pretreatment. Thepercentage of animals remaining tumor-free is plotted. Statisticalsignificance, log rank test P<0.0001.

FIG. 6 demonstrates that chloroquine treatment reduces the developmentof tumors in mice exposed to ionizing radiation in a protocol thatinduces thymic lymphomas. Chloroquine (CHL, 3.5 mg/kg) was given by i.p.injection 24 and 4 hours prior to irradiation with 1.75 Gy in foursuccessive weeks and animals were subsequently observed for thedevelopment of tumors. Statistical significance, log rank test P=0.0012.

FIG. 7 shows tumor incidence in wildtype mice receiving either placeboor CHQ before 3-MC injection. CHQ markedly protects from tumordevelopment.

FIG. 8 shows tumor incidence in ATM-null mice receiving either placeboor CHQ before 3 MC injection. CHQ does not protect from tumordevelopment.

FIG. 9 shows tumor incidence in p53-null mice receiving either placeboor CHQ before 3 MC injection. CHQ does not protect from tumordevelopment.

FIG. 10 shows protection against coat color loss in mice treated withchloroquine or hydroxychloroquine.

DEFINITIONS

The term “animal subject” or “subject” includes humans as well as othermammals. Nonhuman mammals can include veterinary subjects as well asanimal models in which a disorder has been specifically induced tosimulate a human disease. For example, a transgenic mouse model ofstroke is described in Stroke 27:1124-1129 (1996). Severe combinedimmunodeficient mice grafted with human skin are useful as models ofhuman skin cancers, including melanomas.

A subject is at risk of a disorder, if the subject presently lacksclinical symptoms of the disorder, but has a statistically significantlyhigher probability of acquiring the disorder than individuals in thegeneral population. Such risk can be shown by abnormal levels of amarker (e.g., a blood protein) or other sign associated with thedisorder. The risk can also arise through exposure to a condition (e.g.,radiation) associated with a statistically significant increased risk ofthe disorder. The risk can also arise through possession of a geneticvariation associated with the disorder, or first degree relatives havingthe disorder. Levels of a marker are considered abnormal if outside themean plus at least one and preferably at least 2 standard deviations ofthe level of marker in a population of individuals not known to besuffering from a disorder. Likewise a condition is associated withstatistically significant increased risk of the disorder if the numberof individuals in a population of individual exposed to the conditionacquiring the disorder is greater than the number of individuals in apopulation of individuals not exposed to the condition acquiring thedisorder to a statistically significant extent.

Statistical significance is measure at the level of p≦0.05.

A symptom of a disorder means a phenomenon experienced by an individualhaving the disorder indicating a departure from normal function,sensation or appearance.

A sign of a disorder is any bodily manifestation that serves to indicatepresence or risk of a disorder.

The term “treating” includes achieving a therapeutic benefit and/or aprophylactic benefit.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides methods, compositions, and kits for theprevention, prophylaxis and/or treatment of several disorders. Thesedisorders include DNA damage related disorders, cancers, oxidativestress and its many manifestations, stroke, ischemia, andatherosclerosis. Although there is some overlap between these disorders(for example, atherosclerosis is a common cause of ischemia and ischemiaoften gives rise to stroke, the different disorders are not coextensive.For example, atherosclerosis can cause problems by aneurysm as well asischemia. Chloroquine compounds are useful for prevention, prophylaxisand/or treatment of these disorders.

II. Chloroquine Compounds

The term “chloroquine compounds” as used herein means chloroquine-likecompounds, chloroquine and enantiomers, analogs, derivatives,metabolites, pharmaceutically acceptable salts, and mixtures thereof.Examples of chloroquine compounds include, but are not limited to,chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate,chloroquine sulphate, hydroxychloroquine sulphate, and enantiomers,analogs, derivatives, metabolites, pharmaceutically acceptable salts,and mixtures thereof. The term “chloroquine-like compounds” as usedherein means compounds that mimic chloroquine's biological and/orchemical properties.

In a specific embodiment, the invention is practiced with chloroquine.The chemical structure of chloroquine,N⁴-(7-Chloro-4-quinolinyl)-N¹,N¹-diethyl-1,4-pentanediamine or7-chloro-4-(4-diethylamino-1-methylbutylamino) quinoline, is as follows:

Chloroquine (The Merck Index, p. 2220, 1996) is a syntheticallymanufactured drug containing a quinoline nucleus. Suitable synthesistechniques for chloroquine are well known in the art. For example seeU.S. Pat. No. 2,233,970.

As mentioned above, the chloroquine compounds useful herein includechloroquine analogs and derivatives. A number of chloroquine analogs andderivatives are well known. For example, suitable compounds and methodsfor synthesizing the same are described in U.S. Pat. Nos. 6,417,177;6,127,111; 5,639,737; 5,624,938; 5,736,557; 5,596,002; 5,948,791;5,510,356; 2,653,940; 2,233,970; 5,668,149; 5,639,761; 4,431,807; and4,421,920.

Examples of suitable chloroquine compounds include chloroquinephosphate; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline(desmethylchloroquine);7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline(hydroxychloroquine);7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;hydroxychloroquine phosphate;7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline(desmethylhydroxychloroquine);7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino]-6-methoxydihydrochloride quinoline;1-acetyl-1,2,3,4-tetrahydroquinoline;8-[4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-butyryl-1,2,3,4-tetrahydroquinoline;7-chloro-2-(o-chlorostyryl)-4-[4-diethylamino-1-methylbutyl]aminoquinolinephosphate;3-chloro-4-(4-hydroxy-.alpha.,.alpha.′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethylamino)-1-methylbutyl)amino]-6-methoxyquinoline;3,4-dihydro-1 (2H)-quinolinecarboxyaldehyde;1,1′-pentamethylenediquinoleinium diiodide; and 8-quinolinol sulfate,enantiomers thereof, as well as suitable pharmaceutical salts thereof.

Additional suitable chloroquine derivatives include aminoquinolinederivatives and their pharmaceutically acceptable salts such as thosedescribed in U.S. Pat. Nos. 5,948,791 and 5,596,002. Suitable examplesinclude(S)—N₂-(7-Chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine;(R)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-2,N₂,N₂-trimethyl-propane-1,2-diamine;N₃-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,3-diamine;(RS)-(7-chloro-quinolin-4-yl)-(1-methyl-piperidin-3-yl)-amine;(RS)-(7-chloro-quinolin-4-yl)-(1-methyl-pyrrolidin-3-yl)-amine;(RS)—N₂-(7-Chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine;(RS)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine;(S)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine;(R)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine;(RS)-7-chloro-quinolin-4-yl)-(1-methyl-2-pyrrolidin-1-yl-ethyl)-amine;N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-ethane-1,2-diamine;N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-ethane-1,2-diamine;N₃-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,3-diamine;(R)—N₁-(7-chloro-quinolin-4-yl)-N₂,N₂-dimethyl-propane-1,2-diamine;(S)—N₁-(7-chloro-quinoline-4-yl)-N₂,N₂-dimethyl-propane-1,2-diamine;(RS)-(7-chloro-quinolin-4-yl)-(1-methyl-pyrrolidin-2-yl-methyl)-amine;N₁-(7-Chloro-quinolin-4-yl)-N₂-(3-chloro-benzyl)-2-methyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-N₂-(benzyl)-2-methyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-N₂-(2-hydroxy-3-methoxy-benzyl)-2-methyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-N₂-(2-hydroxy-5-methoxy-benzyl)-2-methyl-propane-1,2-diamine;andN₁-(7-chloro-quinolin-4-yl)-N₂-(4-hydroxy-3-methoxy-benzyl)-2-methyl-propane-1,2-diamine;(1S,2S)—N₁-(7-chloro-quinolin-4-yl)-N₂-(benzyl)-cyclohexane-1,2-diamine;(1S,2S)—N₁-(7-chloro-quinolin-4-yl)-N₂-(4-chlorobenzyl)-cyclohexane-1,2-diamine;(1S,2S)—N₁-(7-chloro-quinolin-4-yl)-N₂-(4-dimethylamino-benzyl)-cyclohexane-1,2-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-dimethylamino-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(3-chloro-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(2-hydroxy-4-methoxy-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(3,5-dimethoxy-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-methylsulphanyl-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-diethylamino-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(biphenyl-4-yl)methyl-cyclohexane-1,4-diamine;trans-N₁-(7-chloro-quinolin-4-yl)-N₄-[2-(3,5-dimethoxy-phenyl)-ethyl]-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-methoxy-benzyl)-cyclohexane-1,4-diamine;trans-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-dimethylamino-benzyl)-cyclohexane-1,4-diamine;andtrans-N₁-(7-chloro-quinolin-4-yl)-N₄-(2,6-difluoro-benzyl)-cyclohexane-1,4-diamine.

Chloroquine compounds such as chloroquine may exhibit the phenomena oftautomerism, conformational isomerism, geometric isomerism, and/oroptical isomerism.

The invention covers any tautomeric, conformational isomeric, opticalisomeric and/or geometric isomeric forms of the chloroquine compounds,as well as mixtures of these various different forms.

Chloroquine and hydroxychloroquine are generally racemic mixtures of(−)- and (+)-enantiomers. The (−)-enantiomers are also known as(R)-enantiomers (physical rotation) and 1-enantiomers (opticalrotation). The (+)-enantiomers are also known as (S)-enantiomers(physical rotation) and r-enantiomers (optical rotation). The metabolismof the (+)- and the (−)-enantiomers of chloroquine are described inAugustijins and Verbeke (1993) Clin. Pharmacokin. 24(3):259-69;Augustijins, et al. (1999) Eur. J. Drug Metabol. Pharmacokin.24(1):105-8; DuCharme and Farinotti (1996) Clin. Pharmacokin.31(4):257-74; Ducharme, et al. (1995) Br. J. Clin. Pharmacol.40(2):127-33. Preferably, the (−)-enantiomer of chloroquine is used. Theenantiomers of chloroquine and hydroxychloroquine can be prepared byprocedures known to the art.

The chloroquine compounds may metabolize to produce active metabolites.The used of active metabolites is also within the scope of the presentinvention.

Although an understanding of mechanism is not required for practice ofthe invention, it is believed that one mechanism of action ofchloroquine compounds is to enhance the activity ofAtaxia-Telangiectasia Mutated (ATM) kinase. The agonistic properties ofchloroquine on ATM kinase have been demonstrated (see US Pub. No.20030077661 entitled “ATM Kinase Compositions and Methods,” filed Nov.27, 2003, which is incorporated by reference herein in its entirety).Hence, choroquine-like compounds include compounds that are agonists ofATM kinase. Agonists of ATM kinase include compounds that promote thedissociation of ATM into active monomers and/or compounds that promotephosphorylation of a serine corresponding to the residue 1981 of ATMkinase of SEQ ID NO: 1. Chloroquine compounds may also be effective viaone or other mechanisms that do not involve interaction with ATM.

III. Use of Chloroquine Compounds 1. General

The invention provides methods of prophylaxis or therapeutic treatmentof an animal subject, including a human. The methods generally involvethe administration of effective amounts of chloroquine compoundsincluding chloroquine like compounds for the treatment of one or morethe diseases or disorders described in more detail below. The effects ofthe chloroquine compounds used in the present methods are preferablysystemic, but can be local, or topical depending on the mode ofadministration. Although an understanding of mechanism is not requiredfor practice of the invention, it is believed that chloroquine compoundsact in part by protecting normal cells from radiation or radicals and byinhibiting the cellular damage caused by the radiation or radicals tonormal cells and enhancing the repair process of the normal cells.

2. DNA-Damage Related Disorders

The methods are generally applicable to “DNA damage related disorders.”This term includes cancer, aging, and other disorders caused by damageto DNA due to exposure to carcinogens, toxins, free radicals, likeoxygen radicals, or DNA damaging radiations like ionizing radiation andUV radiation. Cancer and other disorders included in this definition arediscussed individually below. Although an understanding of mechanism isnot required for practice of the invention, it is believed thatadministration of chloroquine compounds prevents DNA damage, inhibitsthe effects of DNA damage, and/or stimulates cellular response to DNAdamage, and that the chloroquine compound acts at least in part throughagonizing ATM and p53.

The p53 gene spans 20 kbp in humans and has 11 exons, 10 of which areprotein coding (see Tominaga et al., 1992, Critical Reviews inOncogenesis 3:257 282, incorporated herein by reference). The geneproduces a 53 kilodalton phosphoprotein that regulates DNA replication.The protein acts to halt replication at the G1/S boundary in the cellcycle and is believed to act as a “molecular policeman,” shutting downreplication when the DNA is damaged or blocking the reproduction of DNAviruses (Kastan et al. Cancer Res 51:6304-6311 (1991); Kuerbitz et al.,Proc Natl Acad Sci USA. 89:7491-7495 (1992); Kastan, Cell 71:587-597(1992), Hartwell et al., Science 266:1821-1828 (1994)).

In one embodiment, chloroquine compounds are used as prophylactics toinhibit side effects of frequent exposure to X-rays in individuals, suchas athletes. This method is useful for other subject populations thatare frequently exposed to DNA damaging radiations, such as X-raytechnicians, police officers, astronauts, and the like. It is known thatexposure to X-rays causes DNA damage. Administration of chloroquinecompounds is contemplated to inhibit the side-effects of frequentexposure to DNA damaging radiations, including inhibit the damage tocells due to damage to DNA.

The prophylactic benefits of chloroquine compounds can be obtained byadministering in advance of exposure to the DNA damaging agent toprovide the enhancing effect in one embodiment. The amount of time priorto the exposure to the DNA damaging agent that the chloroquine compoundis administered can vary from days, hours, to minutes. Also, thechloroquine compounds can be administered during exposure to the DNAdamaging agent or after such exposure. Chloroquine compounds can also beadministered after exposure to the DNA-damaging agent, in which case,the exposure is preferably within one day, a week or a month. In oneembodiment, the effective amount of a chloroquine compound is an amountwhich reduces DNA damage, reduces DNA mutation or increases survival ofcells exposed to a DNA damaging agent when compared to cells exposed tothe same DNA damaging agent and not receiving a chloroquine compound.

3. Cancer

The chloroquine compounds are useful in the prophylaxis, includingprevention, and therapeutic treatment of cancers caused by toxins,carcinogens, DNA damaging radiations, and/or genetic mutations, amongother causes. For example, chloroquine compounds are useful in thetreatment of cancers caused by exposure to toxins and carcinogens likearomatic hydrocarbons, cigarette smoke, acetyl amino fluorine, and MTBE.Also, chloroquine compounds are useful in prophylaxis or therapeutictreatment of cancers caused by DNA damaging radiations like UV andionizing radiation. The ionizing radiation includes both natural andtherapeutic radiation exposures. Examples of ionizing radiations areX-rays for diagnostics and radiation therapy used for tumors. Forexample, chloroquine compounds can be used in the prophylaxis ortreatment of one or more of the following cancers: melanomas, prostatecancer, breast cancer, colon cancer, lung cancer, non-Hodgkin'slymphoma, retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer.

Chloroquine compounds can be used in prophylaxis of subjects at risk ofcancer. Cancer is the uncontrolled, proliferation (growth andmultiplication) of a single type of cell, occurring in any tissue of thebody. Malignant cancers are those that invade surrounding tissues andmetastasize (spread) to other body sites via the blood and lymphaticcirculations. Metastasized cancers usually remain the same type of cellas the initial site of cancer development; for example, if breast cancermetastasizes to a lung, the cancer in the lung consists of breast cells.Benign cancers do not invade other tissues or spread, have a slowergrowth rate than malignant cancers, and in most cases are not fatal. Themethods are particularly useful for prophylaxis or therapeutic treatmentof malignant cancers.

Examples of subjects at risk of cancer include subjects having a primarycancer who are at risk of a second cancer, subjects having been exposedto a carcinogen (e.g., asbestos, benzene) or radiation, includingX-rays, radioactivity, nuclear bombs, dirty bombs, excessive exposure tosun rays, or ultraviolet irradiation, subjects have a genetic variationassociated with cancer, subjects having first degree relatives with acancer known to be hereditary, subjects showing a biochemical markerassociated with cancer (e.g., carcinoembryonic antigen), subjects havingan abnormal cellular morphology (precancerous tissue) associated withdevelopment of cancer (e.g., melanocytic naevi), subjects infected withan oncogenic virus (e.g., certain forms of HPV, HBV, KSHV and EBV) orhelicobacter pylori, and subjects who smoke or engage in excessivedrinking. An exemplary class of subjects at risk of cancer are subjectswho have previously had a melanoma and are risk of developing a furthermelanoma. A further exemplary class of subjects at risk of metastaticmelanoma are subjects having a nonmetastatic melanoma and at least twofamily members with a history of melanoma. Another exemplary class ofsubjects at risk of melanoma are those having congenital melanocyticnaevi. They are brown or black moles which are present at birth, orwhich develop in the first month or so of life.

Genetic risk of cancer can be associated with either homozygous orheterozygous variations in genes. The variations can be present in thegermline (and other cells of a patient) or may appear only inprecancerous or cancerous tissue (i.e., somatic variations). Generally,the most commonly occurring allele in a population is referred to as thewildtype allele, and other less common alleles are referred to asvariant alleles. Some variant alleles in some genes are associated withcancer. Variation can result in cancer either by increased or alteredfunction (e.g., oncogenes) or reduced or lack of function (e.g., tumorsuppressor genes). Variant forms associated with cancer can berecognized by comparing alleles in populations with and without a cancer(usually the individuals in the population with cancer have the sametype for purposes of analysis). Alleles occurring significantly morefrequently in the population having cancer are associated with cancer. Acausative relationship can be conferred by transforming the allele intocells or transgenic animals or knocking out an endogenous allele anddetermining whether the allele causes cancer in the transformed cell oranimal.

Many variations in many genes have already been characterized as beingassociated with cancer. Examples of genes in which variation causesincreased risk of cancer include RB1 retinoblastoma, the WT1 gene forWilms' tumor, the NF1 and NF2 genes for neuroblastomatosis, types 1 and2, the VHL gene for renal cancer tumors association with VonHippel-Lindau disease, the APC gene for adenomatous polyposis coli, andthe BRCAI and II genes for breast and ovarian cancer, PTEN and HRAS-1both associated with breast cancer (see, e.g., Mulligan et al., NatureGenet. 6, 70-74 (1994); Knudson, Cancer Res. 63, 1888-1891 (1989); Mikiet al., Sciences 266, 66-71 (1994); Bronner, Nature 368, 258-261 (1994),Leach, Cell 75, 1215, Lieberman, Amer. J. Gastro. 87, 1085-1093 (1992),Easton, Breast Cancer Research 1, 14-17 (1999), Peto et al., J. Natl.Cancer Inst 91, 943-949 (1999), Kronitiris, N. Engl. J. Med. 329,517-523 (1993); Tirkkonen et al. Cancer Research 57(7), 1222-1227(1997)).

ATM and/or p53 are further examples of genes having variationsassociated with cancer (see, e.g., Garber et al., Cancer Research 51,6094-6097 (1991); Olsen et al., J. Nat. Cancer Inst. 93, 121-127(2001)). As noted these genes are also thought at least in part toeffect the prophylactic and therapeutic benefits of chloroquinecompounds. Optionally, subjects can be screened for variations in thesegenes before commencing treatment. Subjects having wildtype forms of ATMor p53, or heterozygous mutations or homozygous mutations leavingresidual activity of p53 or ATM are preferred for treatment withchloroquine compounds. Subjects having mutations that effectivelyeliminate ATM or p53 function are less preferred for treatment withchloroquine compounds.

For example, retinoblastoma is a cancer affecting young children inwhich tumors develop in the retina of one (unilateral, 75% of cases) orboth (bilateral, 25% of cases) eyes. In all cases, retinoblastoma seemsto be caused by a mutation in the RB1 gene located on chromosome 13,which is responsible for controlling retinal cell division. Althoughretinoblastoma may occur at any age, it most often occurs in youngerchildren, with 80% of cases diagnosed before the age of 5 years.Retinoblastoma presents in both hereditary (40%) and nonhereditary (60%)forms. The hereditary form may manifest as unilateral or bilateraldisease. Most unilateral disease is not hereditary, whereas allbilateral disease is hereditary. For persons carrying the hereditaryform of retinoblastoma, the disease demonstrates with high penetrance(about 90% will manifest disease). Patients with the hereditary type ofretinoblastoma have a markedly increased frequency of additionalmalignancies. The cumulative incidence is about 26+/−10% innonirradiated and 58+/−10% in irradiated patients by 50 years afterdiagnosis of retinoblastoma. Most of the excess cancers wereosteosarcomas, soft tissue sarcomas, or melanomas. The carcinogeniceffect of radiation increased with dose. In irradiated patients, twothirds of the second cancers occur within irradiated tissue and onethird outside the radiation field. Treatment of patients having agenetic variation of the RB1 gene can prevents or reduce the frequencyof both retinoblastomas and/or the additional malignancies.

Methods for determining presence of genetic variations individuals aredescribed in attorney docket 100/1046-20, serial no. [unassigned],“Improvements to Analysis Methods for Individual Genotyping”, filed Feb.24, 2004, U.S. Ser. No. 10/768,788, “Apparatus and Methods for Analyzingand Characterizing Nucleic Acid Sequences”, filed Jan. 30, 2003., andU.S. Ser. No. 10/042,819, “Genetic Analysis Systems and Methods”, filedJan. 7, 2002, and EP 0 730 663, each of which is incorporated byreference.

The presence of risk of cancer in a subject can be determined bybiochemical analysis, cytological examination to detect precancerous orcancerous tissues (e.g., Pap smear), genetic analysis to detect geneticvariation, family history of hereditary cancer, subject history ofexposure to a carcinogen or radiation, or knowledge that the subjectwill be exposed to a carcinogen or radiation. If the risk of cancer isdue to an event that has not yet happened (e.g., occupational exposureto radiation or a carcinogen, or exposure to radiation through treatmentof an existing cancer), chloroquine treatment is preferably administeredbefore the event creating the risk, and or during the event, and/or assoon as possible after the event. Administration can then be continued,preferably at regular intervals, thereafter. If the risk of cancer isdue to an event that has already occurred (e.g., past exposure to acarcinogen or radiation or genetic mutation), chloroquine treatment ispreferably administered as soon as possible after the event, andcontinued at regular intervals thereafter.

In some methods, the subject is monitored to determine response of thesubject to administration of the chloroquine compound. If the subject isat risk of cancer but does not have cancer when the administration isbegun, the subject is monitored to determine whether a cancer develops.The monitoring can be performed by similar techniques to those used inassessing risk of cancer. In addition, cancers or lack thereof can bemonitored by performing scans (e.g., X-rays, CAT scans, MRI) of internalorgans or visuals scans of the skin. If the subject does already have acancer when the administration is begun, the subject is monitored todetermine changes in the cancer responsive to the administration of thechloroquine compound. Preferably, the cancer does not show furtherincrease in size, shrinks in size or disappears. Alternatively,administration can inhibit or stop further growth of the cancer.Monitoring of an existing cancer is performed by visual observation,scanning (e.g., MRI) or biopsy followed by cytological or biochemicalanalysis.

Prophylactic administration of a chloroquine compound can be accompaniedby administration of another chomopreventive agent (e.g., tamoxifen(Nolvadex), Sulindac (Clinoril), or aspirin) or regime designed todecrease the risk of the subject developing cancer. Likewise,therapeutic administration of a chloroquine compound can be accompaniedby administration of a chemotherapeutic agent (e.g., busulfan,cisplatin, cyclophosphamide, methotrexate, daunorubicin, doxorubicin,melphalan, cladribine, vincristine, vinblastine, and chlorambucil) orradiation or other regime designed to treat the tumor.

The chloroquine compounds can be used to prevent secondary cancers,i.e., cancers that are caused by radiation therapy and/or chemotherapyused to treat the primary cancer. For example, the chloroquine compoundsare used to prevent the occurrence of breast cancer in subjectsreceiving radiation therapy for Hodgkin's lymphoma (or other cancers).Also, in these subjects the chloroquine compounds can be used to inhibitthe cellular damage caused by the radiation therapy to normal cells andenhance the repair process of the normal cells. The chloroquinecompounds are also suitable for prevention of the reoccurrence ofcancers in subjects who have had prior incidences of cancer.

The chloroquine compounds can be administered prior to, during, or aftertreatment with radiation. In this embodiment, the beneficial effect ofthe chloroquine compounds is contemplated to be not solely limited to abeneficial effect on pathological skin conditions like skin carcinomasand dermatoses. The use of chloroquine compounds in combination withradiation therapy is contemplated to protect the normal cells andinhibit the cellular damage caused by the radiation therapy to normalcells and enhance the repair process of the normal cells.

The chloroquine compounds can be used in immunosuppressed subjects, liketransplant subjects, who have an increased risk of cancer. Inimmunosuppressed subjects, the chloroquine compounds can be used toprevent cancers. The chloroquine compounds can be used to preventEpstein Barr virus induced lymphoproliferative syndrome.

4. Oxidative Stress

Normal metabolism produces free radical molecules. Free radicals areatoms or molecules which have at least one unpaired electron in theouter orbital. These radicals are the same as generated by externalradiation and include hydrogen peroxide and superoxide. Mitochondria arethe main source of oxygen free radicals under normal conditions. Freeradicals can react with any biological molecule (proteins, lipids,sugars, DNA) altering its structure and often also its function.Therefore living organisms are provided with a rich system ofantioxidant defenses whose main purpose is to prevent the free radicalsattack to other molecules. Antioxidants can also be supplied in the formof nutrition (e.g., phytochemicals in fruit and vegetables). Oxidativestress arises from an imbalance of these radicals and antioxidants as aresult of which unneutralized radicals damage DNA and othermacromolecules.

Oxidative stress contributes to a number of disorders including cancer,coronary disease, atherosclerosis, stroke, cataracts, maculardegeneration, depression, neurodegenerative disease, and prematureaging. Oxidative stress occurs to some extent in everyone. However,levels of oxidative stress that substantially exceed the mean plus astandard deviation in a population of disease free individuals can berecognized as a cause of present or future symptoms, including cancer,coronary disease, atherosclerosis, stroke, cataracts, maculardegeneration, depression, neurodegenerative disease, and prematureaging.

The level of oxidative stress can be quantified by measuring the levelof a series of prostaglandin-like compounds termed isoprostanes. Thesecompounds are formed by perodixation of arachidonic acid. The level ofthese compounds in the plasma or urine is a measure of oxidative stressin a subject (see Morrow, Am. J. Resp. Crit. Care Med. 166, S25-S30(2002)). Subjects having an abnormal level of oxidative stress can berecognized by a level of at least one of isoprostane that exceeds themean plus a standard deviation, preferably, at least two standarddeviations, and more preferably at least five standard deviations of themean level in a population of individuals free of disease. Usually, adetermination of the level of oxidative stress is performed beforecommencing administration of a chloroquine compound. Levels of oxidativestress can be measured at intervals thereafter and compared with abaseline measurement before beginning treatment. Preferably, the leveldecreases after beginning treatment, preferably toward a normal level,or at least the level does not further increase. The subject can also bemonitored for symptoms of oxidative stress, such as maculardegeneration, cataracts or premature aging, cancer, heart disease andimprovement therein responsive to administration of the chloroquinecompound.

The chloroquine compound can be administered in a regime that includesone or more other measures to combat oxidative stress. Such measuresincluding changes in diet, for example, increased intake of fruit andvegetables, and supplementation of the diet with phyotochemicals orantioxidants, such as vitamin B12. Such measures also include increasedexercise, and decreased occupational stress. Such measures also includeadministration of drugs with antioxidant activity such asmethylprednisolone, 21-aminosteroids, 2-methylaminochromans,pyrrolopyrimidines and thiazolidinones. Although an understanding ofmechanism is not required for practice of the invention, it is believedthat administration of a chloroquine compound serves to stimulate thecellular response to DNA damage and promote the repair of the cellsexposed to radicals generated by oxidative stress.

5. Ischemia-Reperfusion

Ischemia refers to a disorder caused by an imbalance between supply anddemand of oxygen to tissue, usually caused by a reduction in blood flowto the tissue. Organs such as the heart and brain are most vulnerable toischemia due to their high extraction of oxygen. Reperfusion is theprocess of restoring blood flow to the tissue. Ischemia and reperfusionresult in different damage to the tissues deprived of oxygen. Thereduction of blood flow decreases the production of high energyphosphates. The energy failure causes membrane depolarization anduncontrolled release of excitatory amino acids, such as glutamate, inthe extracellular space (excitotoxicity). Glutamate acts on varioustypes of receptors, e.g. NMDA and AMPA, eventually causing calciumoverload of neuronal cells. Calcium activates proteolytic enzymes thatbegin to degrade both intracellular and extracellular structures, andother enzymes, i.e. phospholipase A2 and cyclooxigenase, which canproduce free radicals. Neuronal nitric oxide synthase is also calciumdependent and produces nitric oxide, which is able to react withsuperoxide generating the highly reactive radical peroxynitrite.Secondary to ischemia proinflammatory genes are expressed and severalinflammatory mediators are released, such as tumor necrosis factor,interleukin 1β. Adhesion molecules are also expressed and thereforeneutrophils, monocytes and macrophages start to bind the endotheliumcausing microvascular occlusion and cross the vascular wall penetratingin the brain. These inflammatory cells can also produce free radicals.

Restarting blood flow after more than about ten minutes of ischemia istypically more damaging than the ischemia itself because the ischemiasets the stage for oxygen to generate free-radicals. Arachidonic acidconversion to eicosanoids can lead to production of the superoxideradical when oxygen is available. Lipid peroxidation chain reactionsoccur in the membranes of neurons as well as of astrocytes. Free radicaldamage to blood vessels is particularly severe. Ischemia results inlarge amounts of ATP being broken-down to xanthine. Reperfusion allowsthe endothelial enzyme xanthine oxidase to convert xanthine plus oxygento superoxide and uric acid. Liberated iron and zinc ions furtherincrease free radical damage. Superoxide increases the adhesion ofleucocytes to vessel walls. Normally, nitric oxide can combine withsuperoxide to produce peroxynitrite in small amounts, reducing theadhesion. During ischemia, impaired nitric oxide production accompaniesincreased leukocyte adhesion. Reperfusion increases the amount of nitricoxide produced by the endothelial cells. During reperfusion, abnormallyhigh amounts of superoxide converts almost all available nitric oxide toperxoynitrite, which is regarded as the agent causing most of the damageto brain capillary endothelial cells.

Neutrophils which have accumulated in blood vessels due to the ischemiacan also release oxygen-rich free radicals with the availability ofoxygen from reperfusion.

Administration of a chloroquine compound to a subject having or at riskof ischemia is effective in prophylaxis or treatment of tissues damagecaused by ischemia and/or reperfusion, particularly tissue damage ordeath resulting from the generation of free radicals followingreperfusion.

Subjects at risk of ischemia include those having previously had heartdisease, those having elevated biochemical markers of the disease (e.g.,protein. C), those identified as having blockage of blood vessels byangioplasty or MRI imaging, and those undergoing a surgical procedurerequiring temporary obstruction of blood vessels. The presence orabsence and the amount of myocardial damage resulting from prolongedischemia can be assessed by a number of different means, includingpathologic examination, measurement of myocardial proteins in the blood,ECG recordings (ST-T segment wave changes, Q waves), imaging modalitiessuch as myocardial perfusion imaging, echocardiography, contrastventriculography or positron emission tomography (see, e.g., Hanninen etal., Int. J. Bioelectromagnetism No. I Vol. 2 (2000); Alpert, J. Am.College. Cardiol. 36, 959-69 (2000)). Myocardial necrosis results in andcan be recognized by the appearance in the blood of different proteinsreleased into the circulation due to the damaged myocytes: myoglobin,cardiac troponins T and I, creatine kinase, and lactate dehydrogenase.The response of the subject to treatment with a chloroquine can bemonitored by any of these tests. Preferably, the amount of pathologicaldamage or level of a marker associated with the same shows a reducedincrease, does not increase or even is reduced followed administrationof a chloroquine compound relative to a placebo.

In some methods, a chloroquine compound is administered to a subjecthaving or at risk of ischemia in combination with a second agenteffective in prophylaxis of damage resulting from ischemia and/orreperfusion. Such agents include antibodies to adhesion molecules suchas L-selectin, or CD18, tissue plasminogen activator (see EP-B 0 093619), activase, alteplase, duteplase, silteplase, streptokinase,anistreplase, urokinase, heparin, warfarin and coumarin. Additionalthrombolytic agents include saruplase and vampire bat plasminogenactivator.

6. Stroke

Stroke is a sudden loss of brain function resulting from interferencewith the blood supply to the central nervous system. Acute stroke can beclassified either as ischemic (80% of stroke cases), which can befurther classified to extra-cranial embolism and intracranialthrombosis, or a hemorrhagic stroke (20% of stroke cases), which can befurther classified to intracerebral hemorrhage and subarachnoidhemorrhage. Stroke is a leading cause of morbidity and mortality,particularly in the elderly. Its incidence and prevalence sharplyincrease with age: in the United States 72% of the subjects suffering astroke are age 65 and older and up to 88% of stroke deaths occur in thispopulation. Moreover stroke is an important cause of long-termdisability: up to 40% of survivors are not expected to recoverindependence with self-care and 25% become unable to walk independently.Ischemic stroke accounts for 70 to 80% of all strokes and haemorregicstoke accounts for the remainder. Free radicals play an important rolein the pathogenesis of stroke, especially superoxide interact with NO,leading to peroxynitrite formation. Although practice of the inventionis not dependent on an understanding of mechanism, it is believed thatadministration of a chloroquine compound to a subject having or at riskof stroke effects prophylaxis or treatment by mitigating the effects offree radicals, and the response of cells to damage by the free radicals.

Subjects at risk of stroke can be determined by presence of one, andusually at least two of the following risk factors: high blood pressure,heart disease, high cholesterol levels, sleep apnea, previous occurrenceof stroke, smoking, excessive alcohol consumption and excessive weight.Alternatively, transcranial doppler (TCD) testing uses sound waves tomeasure the speed with which blood flows through the large blood vesselswithin the head. The test can detect constriction (narrowing) of bloodvessels as well as blood flow abnormalities related to cerebrovasculardisease. Damage to tissue from stroke can be monitored by MRI and/or bycognitive testing. Monitoring of tissue damage, if any, can be performedfollowing administration of treatment.

Administration of chloroquine can be accompanied by administration ofother agents to treat stroke. These include the same agents discussedfor treating ischemia and oxidative stress as noted above.

7. Atherosclerosis

Atherosclerosis is a process in which deposits of fatty substances,cholesterol, cellular waste products, calcium and other substances buildup in the inner lining of an artery. This buildup is called plaque. Itusually affects large and medium-sized arteries. Some hardening ofarteries often occurs when people grow older. Plaques can grow largeenough to significantly reduce the blood's flow through an artery. Butmost of the damage occurs when they become fragile and rupture. Plaquesthat rupture cause blood clots to form that can block blood flow orbreak off and travel to another part of the body. If either happens andblocks a blood vessel that feeds the heart, it causes a heart attack. Ifit blocks a blood vessel that feeds the brain, it causes a stroke. Andif blood supply to the arms or legs is reduced, it can cause difficultywalking and eventually gangrene. Thus, atherosclerosis as well as beinga disorder in itself, is a risk factor for ischemia and stroke. Insofaras atherosclerosis causes ischemia and/or stroke, it generates freeradicals as these specific diseases and can also be subject toprophylaxis or treatment with a chloroquine compound

Risk factors of atherosclerosis are high blood pressure, high LDL,obesity, high cholesterol levels and smoking. Atherosclerosis can bedetected by MRI or B-mode ultrasound methods (see Tang, Am J CardiacImaging 6:333-339 (1992)). Subjects can be screened for risk factorsbefore commencing treatment. Tissue damage resulting fromatherosclerosis and its sequelae can be monitored following treatment asfor ischemia or stroke.

Administration of a chloroquine compound can be combined with otheragents conventionally used in prophylaxis or treatment ofatherosclerosis. These include antiplatelet agents, lipid loweringagents, bile acid sequestrants, fibrinates, HMG-CoA reductaseinhibitors, nocotinic acid derivatives, and blood pressure loweringagents.

9. Optional Features

Optionally, the methods of the invention are not practiced on subjectswho have presented with a skin carcinoma or epithelioma, like basal cellepithelioma and squamous within one year of beginning treatment with thechloroquine compound. Examples of cancers that are not localized skincarcinomas or epitheliomas include, but are not limited to, melanomas,lymphomas, prostate cancer, breast cancer, colon cancer, lung cancer,retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer. Optionally,the subject has never had a localized skin carcinoma or epithelioma.Optionally, the subject has not suffered from Burkitt's lymphoma, orskin pathologies caused by harmful radiation before administering thechloroquine compound. Optionally, the subject lacks factors predisposingthe subject to Burkitt's lymphomas, such factors being one or more ofmalarial infection, EBV infection (as manifested by antibodies to capsidproteins in the serum detectable by ELISA) and HIV infection.Optionally, the methods are practiced on subjects that are free ofdiseases of the immune system, infectious diseases, neurologicaldiseases, and multidrug resistance (i.e. resistance to multiple drugsfor treatment of the same conditions, such as two anti-cancer drugs, ortwo antibiotics) before commencing administration of the chloroquinecompound. Optionally, the methods are practiced on subjects free ofpsoriasis, malaria, protozoal infections, Alzheimer's disease,Parkinson's disease, lupus erythematosus, rheumatism, hypercalcemia,multiple sclerosis and migraine before administering the chloroquinecompound.

IV. Therapeutic and Prophylactic Benefits

Chloroquine compounds are used as prophylactic agents. For prophylacticbenefit, the chloroquine compound can be administered to a subject atrisk of developing one of the above disorders but not presently showingsymptoms of the disorder being treated. A prophylactic benefit isachieved when a disorder is delayed, reduced in severity or preventedfrom afflicting a subject. A prophylactic benefit can include a resultin which the subject is inflicted with a milder form of the disorderthan in the absence of treatment or the appearance of fewer or nosymptoms of the disorder, or the absence of the disorder in the subjectbeing treated.

Chloroquine compounds can be used for their therapeutic benefits intreating the disorders described above. A therapeutic benefit includeseradication or amelioration of the underlying disorder being treated.For example, in a cancer subject, therapeutic benefit includeseradication, or amelioration of the underlying cancer, or cessation orreduction in its rate of growth. A therapeutic benefit also includes theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the subject, notwithstanding that the subject may still beafflicted with the underlying disorder. For example, administration of achloroquine compound to a subject suffering from cancer providestherapeutic benefit not only when the subject's tumor marker level isdecreased, but also when an improvement is observed in the subject withrespect to other complications that accompany the cancer like pain andpsychiatric disorders. A therapeutic benefit also includes eliminationor reduction of consequences of the underlying disorder, such as thegeneration of free radicals and the resulting damage to macromoleculesand tissue in atherosclerosis, stroke, ischemia and oxidative stress. Atherapeutic benefit can also result when administration of a chloroquinecompound inhibits or prevents further deterioration in the patient'scondition of an existing disorder.

V. Effective Amount

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the chloroquine compound and other optionalactive ingredients are present in an effective amount. The effectiveamounts include doses that partially or completely achieve the desiredtherapeutic, prophylactic, and/or biological effect. The actual amounteffective for a particular application depends on the condition beingtreated and the route of administration. The effective amount for use inhumans can be determined from animal models. For example, a dose forhumans can be formulated to achieve circulating and/or gastrointestinalconcentrations that have been found to be effective in animals.

In some methods, the effective amount includes the dose ranges, modes ofadministration, formulations, and so forth, that have been recommendedor approved by any of the various regulatory or advisory organizationsin the medical or pharmaceutical arts (e.g., FDA, AMA) or by themanufacturer or supplier. Effective amounts of chloroquine can be found,for example, in the Physicians Desk Reference.

In some methods, the daily dosage range of chloroquine, can vary betweenabout 0.1 mg/kg to about 2 gm/kg body weight. The daily dose of achloroquine compound can be less than about 2 gm/kg, less than about 1.5gm/kg, or less than about 1 gm/kg. In some methods, the daily dose of achloroquine compound is more than about 0.5 mg/kg, more than about 500mg/kg, or more than about 1 gm/kg. Some daily dosage ranges of achloroquine compound are about 0.5 mg/lkg to about 50 mg/kg or about 1.0mg/kg to about 10 mg/kg body weight. Some daily doses of chloroquinediphosphate are about 3.5 mg/kg and 7.0 mg/kg.

The dosage can vary depending on the subject being treated. For example,a preferred dosage in mice is 3.5 mg/kg once or twice a day. Theequivalent dosages in monkeys and humans are shown in the Table 1.

TABLE 1 Man (60 kg) Mouse (20 g) Monkey (3.0 kg) Man (60 kg) CHGEquivalent 3.5 mg/kg 0.875 mg/kg 0.292 mg/kg 17.5 mg CHQ 7.0 mg/kg  1.75mg/kg 0.583 mg/kg 35.0 mg CHQ

Preferred dosages ranges in human are from 0.05-1 mg/kg, more preferably0.1 to 0.8 mg/kg, more preferably 0.2-0.6 mg/kg or 0.2 to 0.4 mg/kg. Thedosage can be administered daily, weekly, monthly or bimonthly (everytwo months). In patients whose risk to cancer is occasioned by adistinct event (e.g., exposure to carcinogen or radiation), the dosageis preferably administered daily before, during and/or immediatelyfollowing the event, for an initial period of at least 1 day, 3 days, aweek or a month, and at larger intervals thereafter (e.g., weekly). Forexample, if the risk of exposure is known in advance, an exemplaryregime includes administering the chloroquine compound on the daybefore, the day of exposure and the day after exposure. Anotherexemplary regime comprises administering the chloroquine compound 24hours before the exposure to a known risk of cancer. If the risk ofexposure is not known in advance, an exemplary regime includesadministering the chloroquine compound at least one the day of exposureand the day following exposure. For patients subject to a chronic risk(e.g., through genetic variation), the dosage is preferably administeredweekly, monthly or bimonthly for an indefinite period. The dosage rangecan be lower e.g., 0.05-0.2 mg/kg per day or per week of chloroquine ifa purified (−) enantiomer is used. If hydroxychloroquine is used thedosage range is usually higher than if chloroquine is used.

In some methods, the effective amount of chloroquine is administered atregular intervals, such as every other week, once a week, more than oncea week, or once a day. The dose of chloroquine can be administered onceor more than once a day. In some methods, the effective amount of achloroquine compound is an amount that produces the intended beneficialeffects but does not produce the side-effects associated withchloroquine compounds, like retinoblastoma.

VI. Kits

The invention provides a kit comprising a chloroquine compound packagedin association with instructions teaching a method of using the compoundaccording to one or more of the above-described methods. The kit cancontain the chloroquine compound packaged in unit dosage form.

VII. Routes of Administration and Formulation

The compounds useful in the present invention, or pharmaceuticallyacceptable salts thereof, can be delivered to the subject using a widevariety of routes or modes of administration. Suitable routes ofadministration include, but are not limited to, inhalation, transdermal,oral, rectal, transmucosal, intestinal and parenteral administration,including intramuscular, subcutaneous and intravenous injections.

The chloroquine compounds can be administered topically or systemically.Systemic administration is preferred. In some methods, topicaladministration also has a systemic effect.

The term “pharmaceutically acceptable salt” means those salts whichretain the biological effectiveness and properties of the compounds usedin the present invention, and which are not biologically or otherwiseundesirable. Such salts include salts with inorganic or organic acids,such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitricacid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid,acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid,malic acid, citric acid, tartaric acid or maleic acid. In addition, ifthe compounds used in the present invention contain a carboxy group orother acidic group, it can be converted into a pharmaceuticallyacceptable addition salt with inorganic or organic bases. Examples ofsuitable bases include sodium hydroxide, potassium hydroxide, ammonia,cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine andtriethanolamine.

If necessary, the compounds and useful herein can be administered incombination with other therapeutic agents or regimes as discussed. Thechoice of therapeutic agents that can be co-administered with thecompounds of the invention depends, in part, on the condition beingtreated.

Agents used in accordance with the methods of the invention can beconveniently administered in a pharmaceutical composition containing theactive compound in combination with a suitable carrier. Suchpharmaceutical compositions can be prepared by methods and containcarriers which are well-known in the art. A generally recognizedcompendium of such methods and ingredients is Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippingcottWilliams and Wilkins: Philadelphia, Pa., 2000. Apharmaceutically-acceptable carrier, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, is involved in carrying or transporting the subject compoundfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject.

Examples of materials which can serve as pharmaceutically-acceptablecarriers include sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose, and its derivatives,such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients, such ascocoa butter and suppository waxes; oils, such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;lycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Agents of use in the invention can be administered parenterally (forexample, by intravenous, intraperitoneal, subcutaneous or intramuscularinjection), topically (including buccal and sublingual), orally,intranasally, intravaginally, or rectally, with oral administrationbeing particularly preferred.

For oral therapeutic administration, the composition can be combinedwith one or more carriers and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gums, foods and the like. Also, for oral consumption the activeingredient can be dissolved or suspended in water or other edible oralsolutions. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations can, of course, be varied and can conveniently be betweenabout 0.1 to about 100% of the weight of a given unit dosage form. Theamount of active agent in such therapeutically useful compositions issuch that an effective dosage level is obtained.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. The above listingis merely representative and one skilled in the art could envision otherbinders, excipients, sweetening agents and the like. When the unitdosage form is a capsule, it can contain, in addition to materials ofthe above type, a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials can be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules can be coated with gelatin,wax, shellac or sugar and the like.

For administration orally, the compounds can be formulated as asustained release preparation. Numerous techniques for formulatingsustained release preparations are described in the followingreferences—U.S. Pat. Nos. 4,891,223; 6,004,582; 5,397,574; 5,419,917;5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263;6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379;5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362;5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004;5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177;and WO 98/47491. These references are hereby incorporated herein byreference in their entireties. In a preferred embodiment, the sustainedrelease formulation utilized has an enteric coating.

For administration by inhalation, the active compound(s) can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

A syrup or elixir can contain the active agent, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive components can be incorporated into sustained-releasepreparations and devices including, but not limited to, those relying onosmotic pressures to obtain a desired release profile. Once dailyformulations for each of the active components are specificallyincluded.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation or transcutaneous delivery (forexample subcutaneously or intramuscularly), intramuscular injection or atransdermal patch. Thus, for example, the compounds can be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

The selected dosage level depends on a variety of factors including theactivity of the particular compound of the present invention employed,the route of administration, the time of administration, the rate ofexcretion or metabolism of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compound employed, the age, sex,weight, condition, general health and prior medical history of thesubject being treated, and like factors well-known in the medical arts.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLES Example 1 Radioprotection Assay

HeLa cells were treated with 2 μg/ml of chloroquine for one hour, washedfor one hour, and irradiated at 2 or 6 Gy. Subsequently, 1000 cells wereplated and assessed for colony formation. Table 2 shows that exposure tochloroquine prior to irradiation increased cell survival by 30%.

TABLE 2 Treatment Average Number of Colonies* Std. Dev. 2 Gy 444 19.5Chloroquine + 2 Gy 580 21.2 6 Gy 94.6 10.6 Chloroquine + 6 Gy 129 8.6*Averages were from five individual samples.

To test the possibility that chloroquine activation of ATM may causeradioprotection, C57/BL6 mice were exposed to 8 Gy IR, a dose whichkills approximately 80% of the mice at around two weeks. Death appearsto result from hematopoietic toxicities. The day before total bodyirradiation (TBI), mice were either given an i.p. injection ofchloroquine or chloroquine was added to the drinking water (5 mice—i.p.1.75 mg/kg chloroquine; 5 mice—i.p. 3.5 mg/kg chloroquine; 5 mice—1.75mg/kg chloroquine in drinking water; 5 mice—3.5 mg/kg chloroquine indrinking water). FIG. 1 shows a Kaplan-Meier survival curve indicatingthat a dose of chloroquine prior to the TBI provided significantprotection from death. Significant protection was not observed in ATMdeficient (homozygous) transgenic mice. The experiment was reproducednumerous times and analyses of tissues indicated that the protectiveeffect was due to enhanced recovery of hematopoietic cells (bone marrow,spleen, thymus) following irradiation (FIG. 2). Injection of chloroquineprior to the TBI had no effect on the survival of mice lacking ATM genes(FIG. 3), thus indicating that radioprotection may be dependent on ATM.

Treatment with chloroquine or hydroxychloroquine also providedsignificant protection against loss of coat color in surviving mice.FIG. 10 shows three pairs of mice subject to 8 Gy total bodyirradiation. The two control mice on the left of the figure showsignificant loss of coat color. The pair in the middle which weretreated with chloroquine before exposure to total body irradiation showno significant loss of coat color. The pair on the right treated withhydroxyquinolone show an intermediate extent of protection.

Example 2 Cancer Prevention

Transgenic mice expressing the c-myc oncogene under the control of theimmunoglobulin enhancer (i.e., Eμ-myc mice) develop B-cell lymphomas andleukemias with relatively short latencies. Chloroquine was added to thedrinking water of a cohort of Eμ-myc mice and the mice were observed forthe development of B-cell malignancies. FIG. 4 demonstrates that 100% ofthe control transgenic mice developed malignancies within 100 days ofbirth while 0% of the transgenic mice on chloroquine developed tumors.After ˜120 days, half of the cohort of chloroquine-treated mice weretaken off of chloroquine and the other half were switched to receiving adose of chloroquine by i.p. injection once a week. Within ˜30 days, allof the transgenic mice taken off of the chloroquine had developed tumorswhile none of the mice receiving weekly i.p. injections developedcancer. At ˜10 months of age, these mice on weekly chloroquine remainedcancer-free and appeared healthy and normal.

The carcinogen 3-methylcholanthrene (3-MC) induces soft tissue sarcomasif injected into muscle and skin carcinomas if applied to the skin(Smart et al., Carcinogenesis 7:1669-1675 (1986); Noguchi, et al., Proc.Natl. Acad. Sci. U.S.A 93:11798-11801 (1996); Horak et al., Br. J.Cancer 49:637-644 1984)). This model system has been used to demonstratethat superinduction of p53 after DNA damage (e.g. in a mouse carrying anextra copy of chromosomal DNA containing the p53 gene) protects micefrom the development of cancers induced by chemical carcinogentreatments (Garcia-Cao et al., EMBO J. 21:6225-6235 (2002). Therefore,it was determined whether the protective effect observed in thesestudies could likewise be achieved by biochemically enhancing p53induction. As demonstrated herein, ATM kinase activation by chloroquinedid not induce strand breaks or induce phosphorylation of substratesthat normally get phosphorylated by ATM at the sites of DNA break;however, it did lead to induction and phosphorylation of p53 protein.

In a further experiment, doses of 3.5 mg/kg of chloroquine were given byi.p. injection 24 and 4 hours prior to 3-MC injection in 30 wild type(strain C57B1/6) mice. The occurrence of these tumors was readilyapparent by visual inspection and confirmed by histologic assessment.Results are shown in FIG. 5. Treatment with chloroquine significantlyincreased the percentage of mice surviving tumor free (p=0.0013).

In a further experiment 3-MC was injected into the skin on the leg of amouse once a week for 4 weeks. Three genetic backgrounds were used:wild-type, ATM-null, and p53-null. One half of each cohort of micereceived 3.5 mg/kg of chloroquine (CHQ) via IP injection 24 hours and 4hours prior to each of the four 3-MC administration. The development ofskin carcinomas was followed over time. FIG. 7 shows tumor incidence inmice receiving either placebo or chloroquine prior to 3-MC injection.Chloroquine markedly protected from tumor development. FIG. 8 showstumor incidence in ATM-null mice receiving either placebo or chloroquineprior to 3-MC injection. Chloroquine does not protect from tumordevelopment. FIG. 9 shows tumor incidence in p53-null mice receivingeither placebo or chloroquine prior to 3-MC injection. Again chloroquinedid not protect from tumor development. These results shows that thatthe prophylactic effect of chloroquine is mediated at least in partthrough ATM and p53.

Multiple exposures to non-lethal doses of ionizing radiation can inducethymic lymphomas in C57BL/6 mice (Boniver et al., In Vivo 4(1):41-3(1990)). Using a classical, tumor-inducing protocol (Kaplan and Brown,J. Natl. Cancer Inst., 13, 185-206 (1952)), which consists of fourweekly whole-body exposures of 1.75 Gy each, the effect of chloroquineadministration on thymic lymphoma formation was examined. Chloroquine(3.5 mg/kg) was administered to 4-week old female C57BL/6 mice by i.p.injection 24 hours and 4 hours prior to each of the four doses ofradiation described in the protocol. According to the protocol, tumorswere expected to appear within 4-6 months after the last dose ofirradiation in 90% of control (untreated) mice. FIG. 6 shows the resultsof this analysis. Treatment with chloroquine significantly increased thepercentage of tumor free survival (p<0.0001).

As will be apparent from the foregoing the invention provides forvarious uses. In particular, the invention provides for the use of achloroquine compound in the manufacture of a medicament for prophylaxisor treatment of cancer. The invention further provides for the use of achloroquine compound in the manufacture of a medicament for prophylaxisand treatment of tissue damage resulting from oxidative stress. Theinvention further provides for the use of a chloroquine compound in themanufacture of a medicament for the prophylaxis and treatment of tissuedamage resulting from ischemia and/or reperfusion. The invention furtherprovides for the use of a chloroquine compound in the manufacture of amedicament for the prophylaxis and treatment of tissue damage resultingfrom stroke. The invention further provides for the use of chloroquinein the manufacture of a medicament for the prophylaxis and treatment oftissue damages resulting from atherosclerosis.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made thereto without departing from thespirit or scope of the appended claims.

1.-61. (canceled)
 62. A method for treating atherosclerosis comprisingadministering to a subject in need thereof, an effective amount of achloroquine compound.
 63. The method of claim 62, wherein thechloroquine compound is selected from the group consisting ofchloroquine, chloroquine phosphate, hydroxychloroquine, chloroquinediphosphate, chloroquine sulphate, hydroxychloroquine sulphate, orenantiomers, derivatives, analogs, metabolites, pharmaceuticallyacceptable salts, and mixtures thereof.
 64. The method of claim 63,wherein the chloroquine compound is chloroquine diphosphate.
 65. Themethod of claim 62, wherein the chloroquine compound is administeredintravenously.
 66. The method of claim 62, wherein the chloroquinecompound is administered orally.
 67. The method of claim 62, wherein theadministering is performed at regular intervals for a period of at leastsix months.
 68. The method of claim 62, wherein the chloroquine compoundis administered systemically.
 69. The method of claim 62, wherein thesubject is human.
 70. The method of claim 69, wherein the administrationdosage of the chloroquine compound is 0.05 to 1 mg/kg per day.
 71. Themethod of claim 70, wherein the dosage is 0.2 to 0.6 mg/kg per day. 72.The method of claim 62, wherein the amount of the compound administeredis up to about 10 mg/kg/day.
 73. The method of claim 62, wherein theamount of the compound administered is more than about 0.1 mg/kg/day.74. The method of claim 62, wherein the amount of the compoundadministered is more than about 1.0 mg/kg/day.
 75. The method of claim62, wherein the amount of the compound administered is less than about50 mg/kg/day.
 76. The method of claim 62, wherein the amount of thecompound administered is less than about 10 mg/kg/day.
 77. The method ofclaim 62, wherein the chloroquine compound is administered more thanonce a week.
 78. The method claim 62, wherein the chloroquine compoundis administered daily.
 79. The method of claim 62, wherein thechloroquine compound is formulated in a sustained release formulation.80. The method of claim 62, further comprising administering one or moreadditional active agents to the subject.
 81. The method of claim 80,wherein one or more additional agents are selected from the groupconsisting of antiplatelet agents, lipid lowering agents, bile acidsequestrants, fibrinates, HMG-CoA reductase inhibitors, nocotinic acidderivatives, and blood pressure lowering agents.
 82. The method of claim62, wherein the subject is free of psoriasis, malaria, protozoalinfections, Alzheimer's disease, Parkinson's disease, lupuserythematosus, rheumatism, hypercalcemia, multiple sclerosis.